Mitochondria are prone to environmental, occupational and drug-induced toxicities mediated by oxidative mechanisms. Accumulating evidence indicates that such mitochondrial toxicity contributes to neurodegenerative, cardiovascular and other chronic and age-related diseases. The present research focuses on the delineation of protective functions against such toxicities which are dependent upon the two major thiol antioxidants in mitochondria, thioredoxin-2 (Trx2) and glutathione (mtGSH). Both of these support multiple protective pathways, and both are known to protect in common toxicity models. However, it is not known whether these systems protect against distinct mechanisms in mitochondria. Our preliminary data provide evidence for dependence of toxicity mechanisms upon Trx2 under conditions where GSH was unaffected while another condition showed that GSH/GSSG was affected without a change in Trx2. These results led us to hypothesize that the Trx2 and mtGSH systems support reduction of different proteins and consequently have distinct functions in protecting mitochondria from major mechanisms of oxidative toxicity. In Aim 1, we will use mass spectrometry-based redox proteomic methods to identify physiologic mitochondrial protein substrates for Trx2 and the GSH-dependent mitochondrial glutaredoxin-2 in a doxorubicin-induced toxicity model using a cardiomyocyte cell line. In Aim 2, the role of these Trx2- and mtGSH-dependent protein systems in protecting mitochondria from toxicological insults will be tested. The goal of Aim 3 is to translate these concepts to in vivo studies of doxorubicin cardiotoxicity in mice, using models to experimentally vary the abundance of Trx2 and GSH systems. This research will advance understanding of oxidative toxicities by elucidating details of the Trx2 and GSH pathways in mitochondria and translating these to in vivo models. Given that there are limited means to protect against mitochondrial toxicities, knowledge of these pathways will provide novel therapeutic targets for interventional strategies to protect against disease processes related to chemical-induced mitochondrial dysfunction. PUBLIC HEALTH RELEVANCE: Environmental and occupational exposures can cause toxicity by enhancing the generation of reactive species and by disrupting the signaling processes required for effective regulation of energy production. While much research has focused on the reactive species, little effort has been focused on the signaling processes as a means to protect against toxicity. This is of considerable importance because therapeutic agents which eliminate reactive species could also disrupt normal signaling.